The central concern of clinical chemistry is the qualitative and quantitative determination of specific analytes in samples. Of special concern is the analysis of body fluid samples, such as blood, serum, urine, and so forth. Determination of the presence and/or amount of various analytes, followed by comparison to established parameters determines diagnosis of diseased or abnormal states.
The literature on analytical determination of body fluid samples is an enormous one, as the art has investigated the determination of, e.g., glucose, cholesterol, creatine, sarcosine, urea, and other substances in samples of blood, serum, urine, and so forth.
The early clinical literature taught various non-enzymometric methods for determining analytes. Exemplary of this are the early glucose determination tests taught by Kaplan and Pesce in Clinical Chemistry: Theory, Analysis And Correlation (Mosby, 1984), pages 1032-1042. Such tests include the reduction of copper ions, reaction of copper with molybdate, and so forth. As this reference points out, these methods are insufficiently accurate, due to poor specificity and interference by other analytes. One method described by Kaplan, et al. is the alkaline ferricyanide test. This method involves heating a solution containing glucose in the presence of ferricyanide, at alkaline conditions. The reaction: ##STR1## is accompanied by a change in color from yellow to colorless. Either this decrease in color is measured or the reaction of the colorless ferrocyanide ion with a ferric ion to form the intensely colored precipitate "Prussian Blue" is measured.
Formation of Prussian Blue is an essential part of the invention described herein, so this test will be referred to again, infra.
These early "chelation" type tests became replaced by more specific assays as enzymology became a more developed science. Enzymes are known for their extreme specificity, so via the use of an appropriate enzyme, the skilled artisan could determine, rather easily, whether or not a particular analyte is present, and how much. These enzymatic systems must be combined with indicator systems which, in combination with the enzyme reaction, form a detectable signal. Kaplan describes a glucose-hexokinase system, as well as a glucose oxidase system, and these are fairly well known to the art. They are used in connection with indicator systems such as the "coupled indicators" known as Trinder reagents, or oxidizable indicators such as o-tolidine and 3,3',5,5'-tetramethylbenzidine. In such systems, reaction of the enzyme with its substrate yields a surplus of electrons carried by the enzyme, which are removed by the indicator systems. Color formation follows, indicating presence, absence, or amount of analyte in the sample.
The patent literature is replete with discussions of such systems. A by no means exhaustive selection of such patents include 4,680,259, 4,212,938, 4,144,129 and 3,925,164 (cholesterol oxidase); 4,672,029, 4,636,464, 4,490,465 and 4,418,037 (glucose oxidase); and 4,614,714 (L-glutamic acid oxidase). All of these enzymatic systems "oxidize" their substrates (i.e., the analyte in question) in that they remove electrons therefrom.
Once the analyte loses its electrons, it plays no further part in the determination reaction. As indicated, supra, the electrons may be transferred into a color forming system, such as the Trinder system described in U.S. Pat. No. 4,291,121, or a tetrazolium system, such as is described in, e.g., U.S. Pat. No. 4,576,913.
Indicator systems are not the only means by which the captured electrons may be measured, however. Free electrons produce an electrical current, which can be measured as an indication of analyte. Such systems are described by, e.g.,
Schlapfer, et al., Clin. Chim. Acta 57: 283-289 (1974). These systems employ substances known as "mediators" which remove the electrons from the enzymes. Eventually, the mediators release the electrons as well, producing a measurable current as a result. These mediators can either absorb one, or two electrons per molecule of mediator. Ferricyanide, the preferred mediator described in the Schlapfer reference, picks up one electron per molecule of ferricyanide.
Electron mediators have been known and used in indicator systems in connection with so-called "dye molecules". The 4,576,913 patent, described supra, e.g., teaches the mediator phenazine methosulfate in combination with a tetrazolium salt. It is the latter which serves as the indicator. The use of these mediators enables one to proceed without oxygen. Normally, in a glucose determination reaction, oxygen is necessary to remove electrons from the reduced enzyme. This produces hydrogen peroxide: ##STR2## with the hydrogen peroxide taking part, in the presence of peroxidase, in reactions leading to formation of a color.
It is sometimes undesirable to use oxygen, or aerobic systems, because of various problems inherent in such systems. For example, in these reactions, the reaction is dependent on the partial pressure of O.sub.2 in the atmosphere. In addition, because the O.sub.2 must permeate throughout the entire test medium, the design of such media must be adapted to permit such permeatron. There is interest, then, in indicator systems which are anaerobic, such as those where a mediator is used in connection with the indicator, or electrochemical systems using the mediator alone.
Electrochemical systems, while useful, are not always practical for frequent testing at the present time, and, in terms of home use, individuals who must measure glucose levels daily, are accustomed to systems where a color change is used. Therefore, there exists a need for anaerobic systems which utilize indicator reactions producing a detectable signal, such as a color.
While indicator systems of the type described supra are available, there is a difficulty with these in that the indicator molecules themselves are frequently unstable and do not have long shelf lives. There is therefore an interest in systems which utilize stable molecules which can form a detectable signal.
It will be recalled that Kaplan taught the formation of Prussian Blue in glucose determination, but dismissed it as a viable alternative because of the lack of specificity. Apart from this, the severe conditions under which the reaction are taught to take place are totally unsuitable for enzymatic assays. The reaction Kaplan teaches requires boiling the solution. Enzymes are protein molecules, and inactivation via denaturing is characteristic of what happens when proteins are boiled. Thus, the skilled artisan, seeing the heat parameters of Kaplan would avoid this teaching for enzymatic assays.
Mention of the Prussian Blue system is found in the aforementioned U.S. Pat. No. 4,576,913. This patent teaches a glycerol dehydrogenase which operates in a fashion similar to oxidases in that it teaches removal of two electrons from its substrate molecule. Column 5 of the patent refers to the Prussian Blue system (referred to as "Berlin Blue") as an the indicator.
This patent, however, must be read as a whole, and especially its teaching about the enzyme's operability. Enzymes are extremely pH sensitive, and the enzyme of the Adachi patent is said to operate in a pH range from 6.0 to 10.0, and optimally at 7.0 to 8.5. The teachings, therefore, would suggest to the artisan that since the glycerol dehydrogenase operates at alkaline pHs, the adaptation of the Prussian Blue system to enzyme detection would be at alkaline pHs. However, ferric salts precipitate at alkaline pHs, which would eliminate them from participating in a reaction to form Prussian Blue under the conditions Adachi describes as necessary.
The inventor has now found, quite surprisingly, that enzymatic assays can be performed using the formation of Prussian Blue. These assays do not involve the use of parameters which risk inactivation of the enzyme, such as high heat. The invention is based upon the discovery that, upon addition of sample to a reagent combination containing an enzyme, such as glucose oxidase or cholesterol oxidase, a ferricyanide salt, and a ferric salt buffered at a pH below which the enzyme is expected to be inactivated, the enzyme reactions nonetheless take place, and the buffer does not interfere with the reaction of ferrocyanide and Fe.sup.3+.
Hence it is an object of this invention to provide a reagent composition useful in determining an analyte in a sample, which comprises an enzyme specific for the analyte to be determined, a ferricyanide compound, and a soluble ferric compound, wherein the reagent composition is at a pH below 7.
It is a further object of the invention to provide apparatus, such as test strips which can be used to determine an analyte which incorporate into reagent carriers the above described reagent composition.
Yet another object of this invention is to provide a method for determination of an analyte, comprising contacting a sample to the reagent composition described supra, and measuring formation of Prussian Blue, i.e., the complex of ferrocyanide and ferric ions as a measure of said analyte.
As has been described, this invention is based on the starting and unexpected finding that the ferricyanide/ferric salt system is operable at acidic pH, and under conditions where the enzyme being used would normally be inactive.
How the aforementioned objects of this invention are achieved will be seen in the following Detailed Description of Preferred Embodiments.